Congenital heart diseases account for the highest frequency of human birth defects, affecting l in 100 live births. To gain a better understanding of congenital heart diseases in the human population, the long term goals of this study are to define the molecular mechanisms controlling cardiac cell specification and differentiation in the mouse. The break down in these molecular networks are the ultimate cause of congenital heart diseases, and gaining an understanding of the transcription factors controlling cardiogenesis will facilitate the development of better screens and treatments. Recently, the HAND class of bHLH transcription factors, dHAND and eHAND, were identified and shown to be expressed at the earliest stages of heart formation. Gene disruption experiments of the HAND genes in the chick and mouse show that both of these genes are essential for proper cardiac development. To gain more insight into the biological properties of the HAND genes, transgenic mice expressing LACZ from the dHAND cardiac enhancer, and eHAND knockout mice were generated. These reagents will be used to determine regulatory and functional role that the HAND genes play in cardiogenesis. Specifically, the following questions will be addressed: What are the transcription factors that regulate HAND gene expression? What are the phenotypic defects in mice homozygous for a null eHAND allele? How do the known cardiac transcription factors fit into the cardiogenic molecular pathway? Do these factors interact with each other (directly or indirectly) to construct a normal heart? The completion of this study will result in the identification of novel regulatory factors that act at the earliest stages of cardiogenesis and provide a functional analysis of the HAND genes in heart formation. Taken together, the results of these experiments will provide a comprehensive picture of the function and regulation of the HAND genes. This information will increase understanding of how the molecular pathways that control cardiogenesis are organized, providing greater understanding of the molecular mechanisms controlling heart formation. This understanding is essential for the development of genetic screens and treatments for the many forms of congenital heart disease present in the human population.